Determination of DNA Methylation of Imprinted Genes in &lt;em&gt;Arabidopsis&lt;/em&gt; Endosperm

Rea, Matthew; Chen, Ming; Luan, Shan; Bhangu, Drutdaman; Braud, Max; Xiao, Wenyan

doi:10.3791/2327

Cited by 4 publications

(2 citation statements)

References 16 publications

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“…Four independent triple mutant h1.1‐1 h1.2‐1 h1.3‐1 plants were used in the genetic cross for the bisulfite sequencing assay. Endosperm tissues from seeds of the crosses Col‐0 × RLD and h1.1‐1 h1.2‐1h1.3‐1 × RLD at 8 DAP were isolated, and genomic DNA was extracted as described previously (Rea et al. , 2011).…”

Section: Methodsmentioning

confidence: 99%

Histone H1 affects gene imprinting and DNA methylation in Arabidopsis

Rea¹,

Zheng²,

Chen

et al. 2012

The Plant Journal

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Summary Imprinting, parent-of-origin expression of alleles, plays an important role in regulating development in mammals and plants. DNA methylation catalyzed by DNA methyltransferases plays a pivotal role in regulating imprinting by silencing parental alleles. DEMETER (DME), a DNA glycosylase functioning in the base-excision DNA repair pathway, can excise 5-methylcytosine from DNA and regulate genomic imprinting in Arabidopsis. DME demethylates the maternal MEDEA (MEA) promoter in endosperm, thus resulting in expression of the maternal MEA allele. However, it is not known whether DME interacts with other proteins in regulating gene imprinting. Here we report the identification of histone H1.2 as a DME-interacting protein in a yeast two-hybrid screen and confirmation of their interaction by the in vitro pull-down assay. Genetic analysis of the loss-of-function histone h1 mutant shows that the maternal histone H1 allele is required for DME regulation of the MEA, FWA, and FIS2 imprinting in Arabidopsis endosperm while the paternal allele is dispensable. Furthermore, we show that mutations in histone H1 result in an increase of DNA methylation in the maternal MEA and FWA promoter in endosperm. Our results suggest that histone H1 is involved in the DME-mediated DNA methylation and gene regulation at imprinted loci.

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Section: Methodsmentioning

confidence: 99%

Histone H1 affects gene imprinting and DNA methylation in Arabidopsis

Rea¹,

Zheng²,

Chen

et al. 2012

The Plant Journal

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“…Emasculate flowers and keep them 2 days in the growth chamber while avoiding contact of the exposed pistils with other flowers, then pollinate them (e.g. 11 ). 2.…”

Section: Synchronisation Of Seed Developmentmentioning

confidence: 99%

Efficient and Rapid Isolation of Early-stage Embryos from <em>Arabidopsis thaliana</em> Seeds

Raissig

Gagliardini

Jaenisch

et al. 2013

JoVE

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In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

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Specialized Technologies for Epigenetics in Plants

Xiao

2012

Methods in Molecular Biology

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Plants are excellent systems for discovering and studying epigenetic phenomena, such as transposon silencing, RNAi, imprinting, and DNA methylation. Imprinting, referring to preferential expression of maternal or paternal alleles, plays an important role in reproduction development of both mammals and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA (MEA), a SET domain Polycomb group gene, was the first plant gene shown to be imprinted in endosperm, and its maternal expression is controlled by DNA methylation and demethylation. Recently there has been significant progress in identifying imprinted genes as well as understanding molecular mechanisms of imprinting in plants. Up to date, approximately 350 genes were found to have differential parent-of-origin expression in plant endosperm (Arabidopsis, corn, and rice). In Arabidopsis, many imprinted genes are regulated by the DNA METHYLTRANSFERASE1 (MET1) and the DNA-demethylating glycosylase DEMETER (DME), and/or their chromatin states regulated by Polycomb group proteins (PRC2). There are also maternally expressed genes regulated by unknown mechanisms in endosperm. In this protocol, we describe in detail how to perform a genetic cross, isolate the endosperm tissue from seed, determine the imprinting status of a gene, and analyze DNA methylation of imprinted genes by bisulfite sequencing in Arabidopsis.

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Determination of DNA Methylation of Imprinted Genes in <em>Arabidopsis</em> Endosperm

Cited by 4 publications

References 16 publications

Histone H1 affects gene imprinting and DNA methylation in Arabidopsis

Histone H1 affects gene imprinting and DNA methylation in Arabidopsis

Efficient and Rapid Isolation of Early-stage Embryos from <em>Arabidopsis thaliana</em> Seeds

Specialized Technologies for Epigenetics in Plants

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